Diabetes mellitus is a major and growing health problem in most of the countries. Globally, over the past three decades, the number of individuals with diabetes mellitus has more than doubled, making it one of the foremost public health challenges to all nations. According to estimation by the International Diabetes Foundation, around 592 million people will be affected with diabetes by the year 2040. In India the number of adults suffering from diabetes is expected to increase three-fold, from 19.4 million in 1995 to 57.2 million in 2025. In recent times Type 2 diabetes mellitus (T2DM) and prediabetes are observed to have an increasing incidence among children, adolescents and younger adults. The causes of such epidemic like situation of T2DM are embedded in a very complex group of genetic and epigenetic systems interacting within an equally complex societal framework and intrauterine system which determines environmental influences.
Patients diagnosed with diabetes are susceptible to a range of complications during their life time. Diabetes is addressed to be the leading cause of complications including blindness or retinopathy, amputations, renal dysfunction and neurodegenerative disorders such as diabetic neuropathy. A distinct example of diabetic complications is mirrored in diabetic vascular complications which is the leading cause of end stage renal failure, retinopathy, neuropathies and accelerated atherosclerosis, which accounts for disabilities and high mortality rates in diabetic patients. The etiology of these diabetic complications is poorly understood.
A large body of evidence relating to research investigations have suggested the formation of Advanced Glycation End products (AGE's) to be the leading cause in the development of diabetic complications. For instance AGEs are found in retinal vessels of diabetic patients, and their levels correlate with that in serum as well as with severity of retinopathy. AGE's are a heterogenous group of molecules formed by a series of non-enzymatic reactions between reducing sugars and proteins, especially glucose and glucose derived products which are major glycating agents in hyperglycemic conditions with free amino groups of proteins, lipids, and nucleic acids. The initial product of this reaction is called a Schiff base, which spontaneously rearranges itself into an Amadori, i.e. a deoxufructosylated product, as is the case of the well-known haemoglobin A1c (A1C). A series of subsequent reactions, including successions of dehydrations, oxidation-reduction reactions, and other arrangements lead to the formation of AGEs. Several compounds, e.g., N-carboxymethyl-lysine, N-carboxyethyl-lysine, pentosidine, or methylglyoxal derivatives, serve as examples of well-characterized and widely studied AGEs. (Melpomeni Peppa et al, Clinical diabetes, Vol 21, 4, 2003)
A crucial characteristic of AGE's is their ability for covalent crosslink formation between proteins, resulting in alteration of protein structure and function, as in cellular matrix, basement membranes, and vessel-wall components. Other major features of AGE's relate to their interaction with variety of cell-surface AGE-binding receptors, leading either to endocytosis and degradation or to cellular activation and pro-oxidant, pro-inflammatory events.
Prevention of T2DM is a ‘whole-of-life’ task and requires an integrated approach operating from the origin of the disease. Future research is necessary to better understand the potential role of remaining factors, such as genetic predisposition and maternal environment, to help shape prevention programs. The potential effect on global diabetes surveillance of using glycated haemoglobin (Hb) HbA1c rather than glucose values in the diagnosis of T2DM is well known.
HbA1c/deoxyfructosylated-N1-Val-β-Hb is believed to reflect the glycemic status over the preceding eight to ten weeks. Depending on the estimation method used, HbA1c concentration ranges from 3 to 6.5% of total haemoglobin in normal individuals to as high as 15% in individuals with diabetes. The previous studies have suggested that HbA1c is slowly reversible, and for a given glucose concentration the HbA1c content of red blood cells ultimately reaches an equilibrium value. This suggests that HbA1c does not exactly correlate with the glucose levels in diabetes. HbA1c/deoxyfructosylated-N1-Val-β-Hb, an early product during glycation reaction can undergo carboxymethylation and carboxyethylation
Additionally, glycation is a dynamic reaction; the deoxyfructosylation which is the first and reversible modification of glycation, undergoes various structural rearrangements oxidation, dehydration, condensation, fragmentation, or cyclization leading to the formation of AGEs like carboxymethylation and carboxyethylation. Further, highly reactive dicarbonyls such as glyoxal, methylglyoxal, and 3-deoxyglucose are formed during glycation reaction, which in turn react with proteins and form cross-linked AGE's. Therefore, it is possible that haemoglobin can undergo various AGE modifications referred to as AGE-HbA, as it is one of the long lived blood proteins. The extent of AGE-HbA formation may increase with severity of hyperglycemic condition in diabetes. However, the current diagnostic approaches measure only deoxyfructosylated-N1-Val-β-Hb (HbA1C) and do not measure other AGE-HbA.
The current approaches employed to measure HbA1c levels in human blood include boronate affinity chromatography, HbA1c-specific immunoassays and Ion-exchange based separation. Boronate affinity chromatography detects the presence of cis-diol groups of glycated haemoglobin, therefore levels of Deoxyfructosylation modified haemoglobin which contain the cis-diol group, can be detected, while different other forms of AGEs such as carboxymethylation and carboxyethylation, etc. that are formed during advanced glycation, do not contain the cis-diol group and therefore their levels are unaccounted for in the analysis of glycated haemoglobin. Immunoassays involve the detection of glycation of N-terminus valine residue of the β-chain of the haemoglobin using specific antibodies. By and large the antibodies are raised against only Deoxyfructosylation modification, and therefore the other modifications are not detected. In case of ion-exchange based separation of HbA1C, the amino acid modifications in haemoglobin variants such as Sickle cell haemoglobin, haemoglobin variants D and E also cause a similar change in the net charge as that of the glycated haemoglobin (AGE-HbA) molecule. Thus they can interfere in separation of HbA1c and may lead to over estimation. Additionally, the AGE-HbA may also interfere in analysis as these are quite heterogeneous.
Keeping in mind the crucial role AGE modified haemoglobin serves in diabetic complications and the need to estimate its concentration at regular intervals in diabetics, the present inventors have by employing mass spectrometry characterized the AGE modification of N-terminus valine and other lysine residues of haemoglobin, thus indicating the quantification of AGE-HBA to be a better strategy to assess the glycemic status in diabetic patients.